US10875926B2 - Fully human antibody against human CD 137 - Google Patents
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- US10875926B2 US10875926B2 US15/761,992 US201515761992A US10875926B2 US 10875926 B2 US10875926 B2 US 10875926B2 US 201515761992 A US201515761992 A US 201515761992A US 10875926 B2 US10875926 B2 US 10875926B2
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to a complete human antibody. Specifically, the present invention relates to a fully human antibody against human CD 137 and use thereof.
- CD137 (also known as 4-1BB, TNFRSF9, etc.) is a member of the superfamily of tumor necrosis factor receptors, belonging to type-I transmembrane protein.
- the human CD137 which is a protein comprising 255 amino acids (Uniport: Q07011) of 30 KD, is usually expressed on the cell membrane in the form of a homodimer of 55 KD and will have tripolyzation under the induction of the ligand so as to initiate cell signal conduction.
- CD137L is a member of the superfamily of tumor necrosis factor receptors, belonging to type-II transmembrane protein.
- CD137L is mainly expressed on activated APCs, such as dendritic cells (DC), macrophages and B cells (Pollok, K. E. et al., 1994, Eur. J. Immunol. 24: 367-74); while CD137 may be induced to express after T cells receive the antigen-specific signals (Kwon, B. S. et al., 1989, PNAS 86:1963-67).
- CD137 on T cells has been well proved.
- activation of CD137 signals may induce the proliferation of T cells and the synthesis of cytokines (mainly IFN- ⁇ ), and inhibits the apoptosis of activated T cells, thereby prolonging the life of T cells (D. Laderach et al., 2002, Int.mmunol., 14(10): 1155-67; Croft et al., 2009, Nat Rev Immunol 19:271-285).
- cytokines mainly IFN- ⁇
- a research result showed that CD137 agonist mAb can enhance the killing capability of T lymphocytes in many mice tumor models, leading to an anti-tumor effect (Melero, I. et al., 1997, Nat.
- a fully human antibody against human CD 137 with an active effect is needed for treating and preventing human diseases such as cancer, tumors, infectious diseases and autoimmune diseases.
- the present invention provides a monoclonal antibody specifically binding to CD137 or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.5, a CDR2 having a sequence as set forth in SEQ ID NO.6, and a CDR3 having a sequence as set forth in SEQ ID NO.7; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.10, a CDR2 having a sequence as set forth in SEQ ID NO.11, and a CDR3 having a sequence as set forth in SEQ ID NO.12;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.21, and a CDR3 having a sequence as set forth in SEQ ID NO.22;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.29, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.21, and a CDR3 having a sequence as set forth in SEQ ID NO.22;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.32, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.21, and a CDR3 having a sequence as set forth in SEQ ID NO.22;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.35, and a CDR3 having a sequence as set forth in SEQ ID NO.22;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.21, and a CDR3 having a sequence as set forth in SEQ ID NO.38;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.21, and a CDR3 having a sequence as set forth in SEQ ID NO.41;
- said heavy chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.15, a CDR2 having a sequence as set forth in SEQ ID NO.16, and a CDR3 having a sequence as set forth in SEQ ID NO.17; and
- said light chain variable region comprises a CDR1 having a sequence as set forth in SEQ ID NO.20, a CDR2 having a sequence as set forth in SEQ ID NO.35, and a CDR3 having a sequence as set forth in SEQ ID NO.41.
- the present invention provides a monoclonal antibody specifically binding to CD137 or an antigen binding portion thereof, wherein:
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.4;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.9;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.14;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.19;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.28;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.19;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.31;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.19;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.14;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.34;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.14;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.37;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.14;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.40;
- said heavy chain comprises a variable region having a sequence as set forth in SEQ ID NO.14;
- said light chain comprises a variable region having a sequence as set forth in SEQ ID NO.43.
- the present invention provides a monoclonal antibody specifically binding to CD137 or an antigen-binding portion thereof, wherein the antibody or an antigen-binding portion thereof is a complete antibody, a bispecific antibody, scFv, Fab, Fab′, F(ab′)2 or Fv.
- the present invention provides a single-chain antibody, comprising a VH, a VL and a linker peptide, wherein the VH has a sequence as set forth in SEQ ID NO.4, the VL has a sequence as set forth in SEQ ID NO.9, and the linker peptide has a sequence as set forth in SEQ ID NO.1.
- the present invention provides a single-chain antibody, comprising a VH, a VL and a linker peptide, wherein the VH has a sequence as set forth in SEQ ID NO.14, the VL has a sequence as set forth in SEQ ID NO.23, and the linker peptide has a sequence as set forth in SEQ ID NO.1.
- the present invention provides a pharmaceutical composition, comprising:
- the present invention provides a method for treating cancers in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof.
- the present invention provides a method for combined treatment of a cancer in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof, further comprising administering to the subject a therapeutically effective amount of additional medicaments for treating cancers or implementing other methods for treating cancers.
- the present invention provides a method for treating infectious diseases or autoimmune diseases in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof.
- the present invention provides a method for treating tumors in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof.
- the present invention provides a method for combined treatment of infectious diseases or autoimmune diseases in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof, further comprising administering to said subject a therapeutically effective amount of additional medicaments for treating infectious diseases or autoimmune diseases or implementing other methods for treating infectious diseases or autoimmune diseases.
- the present invention provides a combined treatment method of tumors in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned monoclonal antibody or an antigen-binding portion thereof, further comprising administering to said subject a therapeutically effective amount of additional medicaments for treating tumors or implementing other methods for treating tumors.
- Other methods for treating cancers or tumors described in the present invention include radiotherapy or other approved methods for treating cancers or tumors.
- the present invention also provides an isolated polynucleotide, comprising a nucleotide sequence encoding the amino acid sequences as set forth in SEQ ID NO.4, SEQ ID NO.14, SEQ ID NO.28, and SEQ ID NO.31, or a nucleotide sequence encoding amino acid sequences with a homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
- said isolated polynucleotide comprises nucleotide sequences as set forth in SEQ ID NO.8, SEQ ID NO.18, SEQ ID NO.30, and SEQ ID NO.33, or a nucleotide sequence with a homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
- the present invention further provides an isolated polynucleotide, comprising a nucleotide sequence encoding amino acid sequences as set forth in SEQ ID NO.9, SEQ ID NO.19, SEQ ID NO.34, SEQ ID NO.37, SEQ ID NO.40, SEQ ID NO.43, or a nucleotide sequence encoding amino acid sequences with a homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
- said isolated polynucleotide comprises a nucleotide sequence as set forth in SEQ ID NO.13, SEQ ID NO.23, SEQ ID NO.36, SEQ ID NO.39, SEQ ID NO.42, and SEQ ID NO.44, or a nucleotide sequence as set forth in with a homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
- Beneficial effects of the present invention are as followings: according to the invention, an antibody capable of binding human CD137 protein was obtained through a yeast surface display technology, which is a complete human antibody, and the affinity of the antibody has been greatly enhanced.
- FIG. 1 is a graph showing the results of the binding of purified anti-hCD 137 scFv to hCD137-EGFP cells, where X-axis represents the fluorescence intensity of EGFP, and Y-axis represents the fluorescence intensity of anti-hlg-PE.
- FIG. 2 is a graph showing the results of the specific binding of purified anti-hCD 137 scFv to hCD137-EGFP cells, where X-axis represents the fluorescence intensity of EGFP, and Y-axis represents the fluorescence intensity of anti-hlg-PE.
- FIG. 3 is a graph showing the detection of the binding capacity of anti-CD137 scFv to hCD137 protein.
- FIG. 4 shows the measurement results of the activities of C2 scFv and C14 scFv agonists.
- FIG. 5S shows the yeast staining results by CD137 protein after affinity maturation.
- FIG. 6 is a graph showing the results of purified anti-hCD137 C14# mAb specifically binding to hCD137-EGFP cells, where X-axis represents the fluorescence intensity of EGFP, and Y-axis represents the fluorescence intensity of anti-hlg-PE.
- FIG. 7 is a graph showing the binding capacity of anti-hCD137 antibody to hCD137.
- FIG. 8 is a graph showing the binding capacity of anti-CD137 antibody to Rhesus monkey CD137.
- FIG. 9 is a graph showing the results of anti-CD137 antibody and hCD137L competitively binding to hCD137.
- FIG. 10 shows the detection of the capacity of anti-CD137 C14# mAb stimulating PBMC or CD8+ T cells to secrete IFN- ⁇ .
- FIG. 11 shows the experimental results of anti-CD137 C14# mAb significantly inhibiting the growth of tumors.
- FIG. 12 shows the experimental results of anti-CD137 C14# mAb accelerating the stability.
- FIG. 13 is a graph showing the results of the thermal stability of anti-CD137 C14# mAb detected by DSC method.
- Sequences involved in the present invention include nucleotide sequences and amino acid sequences and have been summarized into a sequence list, attached followed by the specification, and meanwhile, the inventor has submitted the sequence list in a computer readable form.
- antibody refers to any immunoglobulin or complete molecule binding to specific epitope as well as portion thereof.
- the antibodies include, but not limited to, polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, single-chain antibody, and fragments and/or portions of the complete antibody, provided that these fragments or portions remain the antigen binding capabilities of parental antibodies.
- anti-hCD137 antibody refers to a monoclonal antibody, a polyclonal antibody, a single-chain antibody and fragments or portions thereof or functional variants or functional fragments thereof with immune activities, which was capable of specifically binding to human CD137.
- the position of an antibody as used in the present invention is obtained according to the reference on a website, it does not refer to the actual positions of the amino acids in the sequence.
- binding refers to with a purified wild-type antigen, the binding of an antibody with the antigen epitope in the in vitro determination process, preferably in the plasmon resonance determination process (BIAcoreTM, GE-HealthcareTM, Uppsala, Sweden).
- human monoclonal antibody refers to the antibody exhibiting a single binding specificity with variable and constant regions derived from human-type immunoglobulin sequences.
- the amino acid sequence (i.e., from the residue 1 to the residue 186 in Q07011) of the human CD137 extracellular domain was obtained according to the amino acid sequence of human CD137 in the UniprotTM protein database; the amino acid sequence (i.e., from the residue 1 to the residue 186 in F6W5G6) of the monkey CD137 extracellular domain was obtained according to the amino acid sequence (F6W5G6) of rhesus monkey CD137 (RhCD137) in the UniprotTM protein database; the amino acid sequence (i.e., from the residue 104 to the residue 330 in P01857) of the human IgG1-Fc domain was obtained according to the constant region amino acid sequence (P01857) of human immunoglobulin gamma (.gamma.)1 (IgG1) in the UniprotTM protein database; the amino acid sequence (i.e., the residue 98 to the residue 324 in P01868) of mouse IgG1-Fc (muFc) domain was obtained according to the
- the corresponding encoding DNA sequences were designed by using DNAworksTM online tool to obtain the genes of hCD137-Fc, hCD137-muFc and RhCD137-muFc fusion proteins.
- the amino acid sequence (C5MKY7) of enhanced green fluorescent protein (EGFP), the amino acid sequence (Q07011) of human CD137, the amino acid sequence (P20334) of mouse CD137, the amino acid sequence (P41274) of human CD137L, the amino acid sequence (P43489) of human OX40, the amino acid sequence (Q9Y5U5) of human GITR, the amino acid sequence (P26842) of human CD27 were obtained according to the information of the UniprotTM protein database; the corresponding encoding DNA sequences were designed by using DNAworksTM online tool to obtain the above sequences and the genes of EGFP fusion proteins, including the genes of hCD137-EGFP, hCD137L-EGFP, mCD137-EGFP, hOX40-EGFP, hCD
- the recombinant plasmid DNAs were obtained: pcDNA4-hCD137-hFc, pcDNA4-hCD137-muFc, pcDNA4-RhCD137-muFc, pcDNA4-hOX40-EGFP, pcDNA4-hCD137-EGFP, pcDNA4-mCD137-EGFP, pcDNA4-hCD137L-EGFP, pcDNA4-hCD27-EGFP and pcDNA4-hGITR-EGFP.
- HEK293 ATCC, CRL-1573TM cells
- FACS fluorescence activated signal sorting
- pcDNA4-hCD137-Fc, pcDNA4-hCD137-muFc and pcDNA4-RhCD137-muFc were transiently transfected into HEK293 cells for protein production.
- the recombinant expression plasmids were diluted with a FreeStyle293 medium and PEI (polyethylenimine) solution for transformation was added; each group of plasmid/PEI mixture was added into the cell suspension respectively and incubated at 37° C., 10% CO2 and 90 rpm; after 5-6 days, the transiently expressed culture supernatant was collected and purified by Protein ATM affinity chromatography to obtain hCD137-Fc, hCD137-muFc and RhCD137-muFc protein samples for the following examples. The obtained protein samples were subjected to preliminary detection by SDS-PAGE, and the target band can be seen clearly.
- the scFV yeast display library was constructed by cloning the VH and VL genes of the IgM and IgG cDNA from PBMCs of 150 healthy human beings (the linker sequence between the VH and the VL is GGGGSGGGGSGGGGS linker peptide (SEQ ID NO: 1)), with a library volume of 5 ⁇ 10 8 .
- the 10-fold volume of yeast library was resuscitated to induce the expression of the antibody on yeast surface; the yeasts were enriched twice with 100 nM biotinylated hCD137-Fc antigens by magnetic bead sorting, and then further enriched twice with biotinylated hCD137 by flow sorting.
- the enriched yeasts were plated, and monoclones were picked. After amplification and induction of expression, the monoclonal yeasts were analyzed by staining with biotinylated hCD137 or the control antigen hOX40, and the yeast with antigen positive/control yeast negative was regarded as a positive yeast.
- the yeast clones confirmed by FACS were subjected to yeast colony PCR and sequencing.
- the PCR primers were: sequence-F: CGTAGAATCGAGACCGAGGAGA (SEQ ID NO.2); sequence-R: CTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO.3)); sequencing primers were sequence-R. After sequencing, the results were compared and analyzed using BioEditTM software.
- the gene of the single-chain antibody scFv obtained above was fused with the above human IgG1-Fc gene, and then double-digested with HindIII and EcoRI (FermentasTM) and cloned into the commercial vector pcDNA4/myc-HisA.
- the cloning and extraction in small amount of the plasmid were carried out according to the standard operation of Molecular CloningTM.
- the extracted plasmid was transiently expressed in HEK 293 cells and purified through a Protein A column.
- the hCD137-EGFP cells were resuspended in 0.5% PBS-BSA Buffer, and 2 ⁇ g of the above purified anti-hCD137 scFv antibody was added, and the relevant control was set at the same time.
- the negative control was 2 ⁇ g of hIgG 1 protein.
- the secondary antibody was anti-hlg-PE. After staining, it was detected by flow cytometry. In this way, antibodies that bind to the hCD137 antigen on cell surface were identified.
- C2scFv and C14scFv Two antibodies with better properties were obtained: C2scFv and C14scFv.
- the two anti-hCD137 antibodies were able to bind to the hCD137 on cell surface, whereas the negative control was unable to bind to the hCD137 on cell surface.
- the amino acid sequence of heavy chain variable region of C2 scFv is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.5), CDR2 (SEQ ID NO.6), and CDR3 (SEQ ID NO.7), respectively.
- amino acid sequence of light chain variable region of C2 scFv is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.10), CDR2 (SEQ ID NO.11), and CDR3 (SEQ ID NO.12), respectively.
- amino acid sequence of heavy chain variable region of C14 scFv is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.16), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of C14 scFv is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.21), and CDR3 (SEQ ID NO.22), respectively.
- HEK293 cells expressing hCD137-EGFP, hOX40-EGFP, hCD27-EGFP and hGITR-EGFP constructed in Example 1 were resuspended in 0.5% PBS-BSA Buffer and anti-hCD137 C2scFv and C14 scFv protein were added, the negative control was hIgG Fc protein, then the mixture was incubated on ice for 20 min. After washing, secondary antibody anti-hIg-PE (eBioscienceTM) was added and was incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ L of 0.5% PBS-BSA Buffer and detected by flow cytometry. As shown in FIG.
- anti-hCD137 C2scFv and C14 scFv were both able to bind to hCD137-EGFP cells, but unable to bind to several other EGFP cells (hOX40-EGFP-293F, hCD27-EGFP-293F and hGITR-EGFP-293F), showing good capability of specificity.
- hCD137-muFc was diluted to 2 ⁇ g/mL, 100 ⁇ L/well with coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), and then stands overnight at 4° C. After washing, the plates were blocked with 3% BSA-PBS for 1 h at 37° C.
- C2 scFv and C14 scFv antibodies were respectively diluted from 2000 ng/mL and were diluted 2-fold to a total of 11 concentrations, with the diluent (1% BSA-PBS) as a control, and incubated at 37° C. for 2h.
- Goat anti-hIgG-HRP Goat anti-hIgG-HRP-conjugated was added and incubated at 37° C. for 1 h.
- the soluble one-component TMBTM substrate developing solution was added, and the developing was performed in dark at room temperature for 5-10 min. 2N H 2 SO 4 50 ⁇ L/well was added to terminate the color development reaction.
- the OD 450 nm-650 nm values were read on MD SpectraMax Plus 384 microplate ReaderTM, and SoftMax Pro v5.4TM was used for data processing and diagraph analysis, with the results shown in FIG. 3 .
- the binding kinetics of anti-hCD137 C2scFv and C14 scFv antibodies against the recombinant human CD137 were measured by surface plasmon resonance (SPR) process using a BIAcore X100TM instrument.
- Anti-hFc antibody (not cross-identifying mouse Fc) was conjugated on CM5 chip, C2 scFv or C14 scFv was diluted to 5 nM with running buffer and captured as a ligand by the antibody on the chip.
- CD137-muFc was diluted with running buffer to 1000-31.6 nM, diluted twice to a total of 6 concentrations. The injection time was 180 s, the dissociation time was 1800 s and the regeneration time was 60 s.
- the running buffer was HBS-EP+, and the regeneration buffer was 10 mM glycine-HCl (pH 2.0).
- the association rate (K on ) and the dissociation rate (K off ) were calculated using a simple one-to-one LanguirTM binding model (BIAcore Evaluation Software version 3.2TM).
- the equilibrium dissociation constant (K D ) was calculated as the ratio of K off /K on .
- PBMCs Peripheral blood mononuclear cells
- PBMCs peripheral blood concentrated leukocytes of healthy donors by the density gradient centrifugation using human lymphocyte separation fluid (Tianjin Hao YangTM) and seeded into RPMITM complete medium.
- 96-well plates were pre-coated with 50 ⁇ L of 1 ⁇ g/mL anti-CD3 overnight at 4° C.
- mice were coated with 50 ⁇ L of 2 ⁇ g/mL C2 scFv or C14 scFv for 2 h at 37° C., and meanwhile, soluble C2 scFv or C14 scFv with a final concentration of 2 ⁇ g/mL and cross-link (Jackson ImmunoResearch Laboratories: 109-006-008TM) with a final concentration of 2 ⁇ g/mL were added.
- the negative control was RPMITM complete medium.
- the amount of PBMCs was 2 ⁇ 10 5 /well, the cells were cultured for five days and then the supernatant was taken. As shown in FIG.
- the level of IFN- ⁇ in the supernatant was detected by the IFN- ⁇ ELISATM into RPMITM detection kit (EioscienceTM) and the proliferation of T cells was detected by the BrdUTM staining kit (Roche: 11647229001). It can be seen that C2 scFv and C14 scFv have good activity in activating PBMCs and promoting the proliferation of T cells under both coating administration mode and cross-link administration mode, while the agonist activity of C14 scFv is slightly stronger than that of C2 scFv.
- PBMCs Peripheral blood mononuclear cells
- PBMCs peripheral blood concentrated leukocytes of healthy donors (4# and 5#) by the density gradient centrifugation using human lymphocyte separation fluid (Tianjin Hao YangTM) and seeded into RPMITM complete medium.
- 96-well plates were pre-coated with 50 ⁇ L of 1 ⁇ g/mL anti-CD3 overnight at 4° C.
- Experimental groups were coated with 50 ⁇ L of 2 ⁇ g/mL C14 scFv and cross-link (Jackson ImmunoResearch Laboratories: 109-006-008TM) with a final concentration of 2 ⁇ g/mL.
- the negative control was RPMITM complete medium.
- the amount of PBMCs was 2 ⁇ 10 5 /well, the cells were cultured for five days and then the supernatant was taken. As shown in FIG. 4 , the level of IFN- ⁇ in the supernatant of 5# PBMC ( FIG. 4 ) and the level of IFN- ⁇ in the supernatant of 4# PBMC ( FIG. 4 ) were detected by the IFN- ⁇ ELISATM detection kit (eioscienceTM), it can be seen that C14 scFv could increase the level of the activated PBMC secreting IFN- ⁇ .
- cMyc-BBXhoI GCCAGATCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTAGAATTCC G (SEQ ID NO.25) as primers.
- the resulting PCR products were digested with NheI and BglII (FermentasTM) to construct a recombinant plasmid.
- NheI and BglII FermentasTM
- a random mutation PCR product of scFv was obtained by error prone PCR with reference to the method of Ginger et al. (2006) NatProtocl (2): 755-68.
- the primers used were ep-F: TAATACGACTCACTATAGGG (SEQ ID NO.26) and ep-R: GGCAGCCCCATAAACACACAGTAT (SEQ ID NO.27).
- the resulting PCR products were purified by the GeneJET DNA purification KitTM from Fermentas and then precipitated in ethanol to a concentration greater than 1 ⁇ g/ ⁇ L.
- the remaining operation steps refer to the method of Ginger et al. (2006) Nat Protocl (2): 755-68 to obtain a yeast library with mature affinities by virtue of yeast electrical conversion and in vivo recombination method.
- the affinity-matured yeast library obtained above was subjected to two rounds of fluorescence-activated cell sorting with 10 nM and 1 nM hCD137-Fc protein, and the resulting yeast products were plated and subjected to monoclonal identification.
- the yeast monoclones with improved affinity were identified by flow staining, with the results of yeast staining shown in FIG. 5 .
- yeast clones confirmed by FACS were subjected to yeast colony PCR and sequencing, the method as above.
- the results of sequence analysis were shown in the following table:
- the amino acid sequence of the human IgG4 constant region was obtained based on the amino acid sequence (P01861) of the constant region of the human immunoglobulin gamma( ⁇ )4 (IgG4) in the UniprotTM protein database.
- the corresponding encoding DNA sequence was designed by using DNAworksTM online tool to obtain the gene of the human IgG4 constant region.
- VH sequence of the C14 heavy chain variable region obtained through screening was spliced with the gene sequence of the human IgG4 constant region, and the spliced genes were synthesized, and double-digested with HindIII and EcoRI (FermentasTM) and subcloned into the vetor pcDNA4/myc-HisA, to obtain pcDNA4-C14HC.
- the amino acid sequence of the human lambda light chain constant region was obtained based on the amino acid sequence (AOM8Q6) of the constant region of the human immunoglobulin lambda ( ⁇ ) in the UniprotTM protein database.
- the corresponding encoding DNA sequence was designed by using DNAworksTM online tool to obtain the gene of the human lambda ( ⁇ ) light chain constant region.
- VL sequence of the C14 light chain variable region obtained through screening was spliced with the gene sequence of the human lambda ( ⁇ ) light chain constant region, and the spliced genes were synthesized, and double-digested with HindIII and EcoRI (FermentasTM) and subcloned into the vetor pcDNA4/myc-HisA, to obtain pcDNA-C14LC.
- Plasmid extraction of the heavy and light chain plasmids obtained above was carried out using the plasmid extraction kit (PL14) supplied by AidLabTM.
- the recombinant constructed light chain and the heavy chain plasmids were co-transfected into HEK293 cells to carry out the antibody expression, transiently cultured for 5-6 days, and then the culturing supernatant was collected, and purified through a Protein ATM affinity chromatography method to obtain an anti-hCD137 antibody: C14mAb.
- the scFv-type antibody with matured affinity was formatted into an IgG-type antibody according to the same method, and a series of anti-CD137 14# mAb variants were obtained, as shown in the following table.
- amino acid sequence of heavy chain variable region of anti-CD137 14# H54H57 mAb is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.29), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of anti-CD137 14# H54H57 mAb is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.21), and CDR3 (SEQ ID NO.22), respectively.
- amino acid sequence of heavy chain variable region of anti-CD137 14# H32 mAb is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.32), CDR2 (SEQ ID NO.16), CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of anti-CD137 14# H32 mAb light chain variable region is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.21), and CDR3 (SEQ ID NO.22), respectively.
- amino acid sequence of heavy chain variable region of anti-CD137 14# L50 mAb is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.16), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of anti-CD137 14# L50 mAb is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.35), and CDR3 (SEQ ID NO.22), respectively.
- amino acid sequence of heavy chain variable region of anti-CD137 14# L95mAb is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.16), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of anti-CD137 14# L95mAb is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.21), and CDR3 (SEQ ID NO.36), respectively.
- amino acid sequence of heavy chain variable region of anti-CD137 14# L93L95mAb is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.16), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of anti-CD137 14# L93L95mAb is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.21), and CDR3 (SEQ ID NO.41), respectively.
- amino acid sequence of heavy chain variable region of anti-CD137 14# mAb new is:
- the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.15), CDR2 (SEQ ID NO.16), and CDR3 (SEQ ID NO.17), respectively.
- amino acid sequence of light chain variable region of anti-CD137 14# mAb new is:
- amino acids in frame were mutation sites, the amino acids corresponding to the underlined parts are CDR1 (SEQ ID NO.20), CDR2 (SEQ ID NO.35), and CDR3 (SEQ ID NO.41), respectively.
- HEK293 cells expressing hCD137-EGFP, hOX40-EGFP, hCD27-EGFP and hGITR-EGFP constructed in Example 1 were resuspended in 0.5% PBS-BSA Buffer and anti-hCD137 C14 mAb protein was added, the negative control was hIgG Fc protein, then the mixture was incubated on ice for 20 min. After washing, a secondary antibody anti-hIg-PE (eBioscienceTM) was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ L 0.5% PBS-BSA Buffer and detected by flow cytometry. Results were shown in the figure. As shown in FIG.
- anti-hCD137 C14 mAb was able to bind to hCD137-EGFP cells, but unable to bind to several other EGFP cells (hOX40-EGFP-293F, hCD27-EGFP-293F and hGITR-EGFP-293F), showing good capability of specificity.
- hCD137-muFc was diluted to 2 ⁇ g/mL with coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), 100 ⁇ L/well, overnight at 4° C. After washing, the plates were blocked with 3% BSA-PBS for 1 h at 37° C.
- C14 mAb and variants thereof were respectively diluted from 2000 ng/mL and were diluted 2-fold to a total of 11 concentrations, with the diluent (1% BSA-PBS) as a control, and incubated for 2h at 37° C.
- Goat anti-hIgG-HRP Goat anti-hIgG-HRP conjugated
- the soluble one-component TMB substrate developing solution was added, and the developing was performed in dark at room temperature for 5-10 min. 2N H 2 SO 4 50 ⁇ L/well was added to terminate the color development reaction.
- the OD 450 nm-650 nm values were read on MD SpectraMax Plus 384 microplate ReaderTM, and SoftMax Pro v5.4TM was used for data processing and diagraph analysis, with the results shown in FIG. 7 .
- the binding of the antibody and the antigen was not affected when H54 and H57 sites of the heavy chain were mutated at the same time, and meanwhile, the affinity between the antibody and the antigen did not increase; as shown in FIG. 7B , the mutation of the light chain could increase the affinity between the antibody and the antigen, especially when L93 and L95 sites were mutated at the same time. It can be seen from FIG. 7C that, the affinities of anti-CD137 14# L93L95mAb and anti-CD137 14# mAb new with hCD137 were similar to each other.
- the binding kinetics of anti-hCD137 antibody against the recombinant human CD137 were measured by surface plasmon resonance (SPR) process using a BIAcore X100 TM instrument.
- Anti-hFc antibody (not cross-identifying mouse Fc) was conjugated on CM5 chip, CD137-muFc was diluted to 5 nM with running buffer and captured as a ligand by the antibody on the chip.
- CD137-muFc was diluted with running buffer to 1000-31.6 nM (C14Mab) or 100-3.16 nM (C14 Mab new), diluted twice to a total of 6 concentrations.
- the injection time was 180s, the dissociation time was 1800s and the regeneration time was 60s.
- the running buffer was HBS-EP+, and the regeneration buffer was 10 mM glycine-HCl (pH 2.0).
- the association rate (K on ) and the dissociation rate (K off ) were calculated using a simple one-to-one one LanguirTM binding model (BIAcore Evaluation Software version 3.2TM).
- the equilibrium dissociation constant (KD) was calculated as the ratio of K off /K on .
- the measured binding affinities of anti-hCD137 antibodies were seen in Table 3.
- RhCD137-muFc was diluted to 5 ⁇ g/mL 100 ⁇ L/well, with coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), overnight at 4° C. After washing, the plates were blocked with 3% BSA-PBS for 1 h at 37° C.
- Anti-hCD137 14# mAbnew antibodies were respectively diluted from 2000 ng/mL and were diluted 3-fold, with the diluent (1% BSA-PBS) as a control, and incubated for 2 h at 37° C. Goat anti-hIgG-HRP conjugated was added and incubated for 1 h at 37° C.
- the soluble one-component TMB substrate developing solution was added, and the developing was performed in dark at room temperature for 5-10 min. 2N H 2 SO 4 50 ⁇ L/well was added to terminate the color development reaction.
- the OD 450 nm-650 nm values were read on MD SpectraMax Plus 384 microplate ReaderTM, and SoftMax Pro v5.4TM was used for data processing and diagraph analysis, with the results shown in FIG. 8 , in which it can be seen that anti-hCD137 14# mAb new may bind to CD137 of rhesus monkey.
- the staining was detected by a flow cytometer, with results shown in FIG. 9 , wherein X-axis represents the fluorescence intensity of EGFP, Y-axis represents the fluorescence intensity of PE. It can be seen from the results that, anti-CD137 14# mAbnew here did not block the binding between CD137L and CD137 protein.
- PBMCs Peripheral blood mononuclear cells
- PBMCs peripheral blood concentrated leukocytes of healthy donors by the density gradient centrifugation using human lymphocyte separation fluid (Tianjin Hao YangTM) and seeded into RPMITM complete medium.
- 96-well plates were pre-coated with 50 ⁇ L of 10 ⁇ g/mL anti-CD3 and 0.5 ⁇ g/mL soluble anti-CD28 overnight at 4° C.
- Experimental groups were coated with 50 ⁇ L of 2 ⁇ g/mL C14 ScFv or C14 mAb and cross-link (Jackson ImmunoResearch Laboratories: 109-006-008) with a final concentration of 2 ⁇ g/mL, the negative control was RPMITM complete medium.
- the amount of PBMCs was 2 ⁇ 10 5 /well, the cells were cultured for five days and then the supernatant was taken.
- the level of IFN- ⁇ in the supernatant of PBMC was detected by the IFN- ⁇ ELISA detection kit (eBioscienceTM) and the results were shown in FIG. 10A . It can be seen from the results that anti-CD137 14# ScFv and mAb both could upregulate the level of IFN- ⁇ secreted by the activated PBMC.
- PBMCs Peripheral blood mononuclear cells
- PBMCs peripheral blood concentrated leukocytes of healthy donors by the density gradient centrifugation using human lymphocyte separation fluid (Tianjin Hao YangTM) and seeded into RPMITM complete medium.
- CD8+ T cells were isolated from PBMC by using the magnetic bead separation kit (Miltenyi Biotec:130-096-533), according to the method in the specification. They were weighed and resuspended in RPMITM complete medium, with a concentration of 2 million/mL. The isolated CD8+ T cells were stimulated with 1 ⁇ g/mL anti-CD3 and 0.2 ⁇ g/mL anti-CD28 to be activated.
- the NOD-SCID mouse tumor models implanted with tumor cells PC-3 and human PBMCs were used to evaluate the in vivo efficacy of anti-CD137 antibody.
- Mice were injected subcutaneously (SC) with PC-35 ⁇ 106 (ATCCCRL-1435TM) and human peripheral blood mononuclear cells 2.5 ⁇ 10 6 (PBMCs) on day 0 and injected intraperitoneally with 1 mg/kg C14 mAb on day 0 and day 7, PBS was used as the negative control.
- Tumor formation was observed twice a week and the length diameters and short diameters of the tumors were measured with a vernier caliper. The tumor volume was calculated and the tumor growth curve was plotted. The results were shown in FIG. 11 , and it could be seen that the antibody C14 mAb could significantly inhibit the tumor growth.
- Fig. A is a graph of antibody concentration over time, and it can be seen that the concentrations of samples collected at different time points did not change significantly;
- Fig. B is the percentage of the antibody dimer over time, and it can be seen that as time increases, the proportion of the antibody dimer was slightly reduced, and no aggregate was formed.
- the thermal stability of anti-CD137 14# mAb was detected by DSC method.
- the scanning results of a single buffer solution and a buffer solution containing protein were collected.
- the anti-CD137 14# mAb protein was diluted to 1 mg/mL (PBS buffer). Data was collected under the following conditions: the DSC was set to scan at 10-110° C. at the scanning speed of 100° C./h, and there was equilibrium of 15 minutes before each scanning. The volume of DSC sample chamber was 0.5 mL. After collection of the scanning results of the buffer and the protein, the scanning result of the protein can be subtracted from the scanning result of the buffer. The concentration of protein in the sample was obtained to correct the concentration in each scanning, thus obtained the Tm value of anti-CD137 14# mAb, with the results shown in FIG. 13 , from which it can be seen that the Tm value of anti-CD137 14# mAb was 69.70° C.
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CN111511762A (zh) | 2017-08-21 | 2020-08-07 | 天演药业公司 | 抗cd137分子及其用途 |
TWI701259B (zh) * | 2017-11-09 | 2020-08-11 | 大陸商上海懷越生物科技有限公司 | 4﹘1bb抗體及其製備方法和應用 |
WO2019104716A1 (fr) * | 2017-12-01 | 2019-06-06 | Adagene Inc. | Méthodes d'utilisation du ligand cd137 comme biomarqueur pour le traitement avec un anticorps anti-cd137 |
CA3089260A1 (fr) * | 2018-01-22 | 2019-07-25 | Jiangsu Hengrui Medicine Co., Ltd. | Anticorps anti-4-1bb, fragment de liaison a l'antigene de celui-ci et utilisation medicale associee |
WO2019148445A1 (fr) | 2018-02-02 | 2019-08-08 | Adagene Inc. | Anticorps activables dépendant de la précision/du contexte, et leurs procédés de fabrication et d'utilisation |
GB201811408D0 (en) * | 2018-07-12 | 2018-08-29 | F Star Beta Ltd | CD137 Binding Molecules |
WO2020077635A1 (fr) * | 2018-10-19 | 2020-04-23 | 苏州丁孚靶点生物技术有限公司 | Anticorps anti-cd137 et ses applications |
BR112021010402A2 (pt) * | 2018-11-30 | 2021-08-24 | Abl Bio Inc. | Anticorpos biespecíficos anti-pd-l1/anti-4-1bb e uso dos mesmos |
CA3125451A1 (fr) * | 2019-01-02 | 2020-07-09 | Qlsf Biotherapeutics Inc. | Anticorps agonistes de cd137 et utilisations associees |
JP2022518441A (ja) * | 2019-01-16 | 2022-03-15 | コンパス セラピューティクス リミテッド ライアビリティ カンパニー | ヒトcd137に結合する抗体の製剤およびその使用 |
AU2020263959A1 (en) | 2019-04-24 | 2021-11-25 | Heidelberg Pharma Research Gmbh | Amatoxin antibody-drug conjugates and uses thereof |
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CN112279915A (zh) * | 2019-07-25 | 2021-01-29 | 苏州丁孚靶点生物技术有限公司 | Icos抗体、基因、载体、宿主细胞和icos拮抗剂 |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000029445A1 (fr) | 1998-11-17 | 2000-05-25 | Lg Chemical Limited | Anticorps humanise specifique a la proteine humaine 4-1bb et composition pharmaceutique le comprenant |
WO2005035584A1 (fr) | 2003-10-10 | 2005-04-21 | Bristol-Myers Squibb Company | Anticorps entierement humains agissant contre la 4-1bb humaine (cd137) |
WO2006088447A1 (fr) | 2005-02-15 | 2006-08-24 | Gtc Biotherapeutics, Inc. | Anticorps anti-cd137 en tant qu'agent dans le traitement de cancers et variante de glycosylation dudit anticorps |
CN1867585A (zh) | 2003-10-10 | 2006-11-22 | 布里斯托尔-迈尔斯·斯奎布公司 | 抗人4-1bb(cd137)的完全人抗体 |
JP2013544756A (ja) | 2010-09-09 | 2013-12-19 | ファイザー・インク | 4−1bb結合分子 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030118588A1 (en) * | 1999-05-22 | 2003-06-26 | Linda Diehl | Induction of anti-tumor CTL immunity through in vivo triggering of 4-1BB and/or CD40 |
WO2004010947A2 (fr) * | 2002-07-30 | 2004-02-05 | Bristol-Myers Squibb Company | Anticorps humanises contre le 4-1bb humain |
US20070253961A1 (en) * | 2004-06-09 | 2007-11-01 | Ulsan Industrial Education Foundation | Pharmaceutical Composition Comprising the Anti-4-1Bb Antibody for Treating or Preventing Rheumatoid Arthritis |
-
2015
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- 2015-09-22 EP EP15904340.5A patent/EP3354661B1/fr active Active
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- 2015-09-22 US US15/761,992 patent/US10875926B2/en active Active
-
2020
- 2020-06-29 HR HRP20201022TT patent/HRP20201022T1/hr unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000029445A1 (fr) | 1998-11-17 | 2000-05-25 | Lg Chemical Limited | Anticorps humanise specifique a la proteine humaine 4-1bb et composition pharmaceutique le comprenant |
CN1357009A (zh) | 1998-11-17 | 2002-07-03 | 株式会社Lg化学 | 人源化的特异于人4-1bb的抗体和含有它的药物组合物 |
WO2005035584A1 (fr) | 2003-10-10 | 2005-04-21 | Bristol-Myers Squibb Company | Anticorps entierement humains agissant contre la 4-1bb humaine (cd137) |
CN1867585A (zh) | 2003-10-10 | 2006-11-22 | 布里斯托尔-迈尔斯·斯奎布公司 | 抗人4-1bb(cd137)的完全人抗体 |
JP2007532095A (ja) | 2003-10-10 | 2007-11-15 | ブリストル−マイヤーズ スクイブ カンパニー | ヒト4−1bb(cd137)に対する完全ヒト抗体 |
WO2006088447A1 (fr) | 2005-02-15 | 2006-08-24 | Gtc Biotherapeutics, Inc. | Anticorps anti-cd137 en tant qu'agent dans le traitement de cancers et variante de glycosylation dudit anticorps |
JP2013544756A (ja) | 2010-09-09 | 2013-12-19 | ファイザー・インク | 4−1bb結合分子 |
Non-Patent Citations (11)
Title |
---|
Chu et al., International Journal of Molecular Sciences 2019, 20, 1822; pp. 1-17. (Year: 2019). * |
Croft, Michael; The Role of TNF Superfamily Members in T-Cell Function and Diseases; Nature Reviews Immunology; Macmillan Publishers Ltd.; vol. 9; Apr. 2009; pp. 271-285. |
Fisher, Timothy S. et al; Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity; Cancer Immunology, Immunotherapy, vol. 61, No. 10, Mar. 11, 2012, pp. 1721-1733, XP55391951, Berlin/Heidelberg, ISSN 0340-7004. |
Kwon, Byoung S. et al; cDNA Sequences of Two Inducible T-Cell Genes; Proc Natl Acad Sci USA; vol. 86, pp. 1963-1967; Mar. 1989; Immunology. |
Laderach, Diego et al; 4-1BB Co-Stimulation Enhances Human CD8+ T Cell Priming by Augmenting the Proliferation and Survival of Effector CD8+ T Cells; Intl Immunology, vol. 14, No. 10, pp. 1155-1167; Japanese Soc for Immunology; Jul. 2002. |
Melero, Ignacio et al; Monoclonal Antibodies Against the 4-1BB T-Cell Activation Molecule Eradicate Established Tumors; Nature Medicine, vol. 3, No. 6, Jun. 1997, Nature Publishing Grp; pp. 682-685. |
PCT/CN2015/090226 International Search Report dated Jun. 22, 2016. |
Pollok, Karen E. et al; 4-1BB T-Cell Antigen Binds to Mature B Cells and Macrophages, and Costimulates Anti-μ-Primed Splenic B Cells; European J Immunol, 1994, 24:367-374; VCH Verlagsgesellschaft mbH 0014-2980/94/0202-0367. |
Qi et al., Nature Communications 201910:2141; doi.org/s41467-019-10088-1; nature.com/naturecommunications; pp. 1-11. (Year: 2019). * |
Shi, Wenyin et al; Augmented Antitumor Effects of Radiation Therapy by 4-1BB Antibody (BMS-436492) Treatment; Anticancer Research, 26: 3445-3454; 2006; 0250-7005/2006. |
TIMOTHY S. FISHER, CRIS KAMPERSCHROER, THEODORE OLIPHANT, VICTORIA A. LOVE, PAUL D. LIRA, REGIS DOYONNAS, SIMON BERGQVIST, SANGITA: "Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity", CANCER IMMUNOLOGY, IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 61, no. 10, 1 October 2012 (2012-10-01), Berlin/Heidelberg, pages 1721 - 1733, XP055391951, ISSN: 0340-7004, DOI: 10.1007/s00262-012-1237-1 |
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JP6845846B2 (ja) | 2021-03-24 |
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